Global warming intensifies the interference competition by a poleward-expanding invader on a native dragonfly species

Rapid climate warming has boosted biological invasions and the distribution or expansion polewards of many species: this can cause serious impacts on local ecosystems within the invaded areas. Subsequently, native species may be exposed to threats of both interspecific competition with invaders and temperature rises. However, effects of warming on interspecific interactions, especially competition between invader and native species remains unclear. To better understand the combined threats of biological invasions and warming, the effect of temperature on competitive interactions between two dragonfly species, the expanding Trithemis aurora from Southeast Asia and the Japanese native Orthetrum albistylum speciosum were assessed based on their foraging capacity. Although the stand-alone effect of temperature on foraging intake of the native dragonfly was not apparent, its intake significantly decreased with increasing temperatures when the invader T. aurora was present. Such reductions in foraging might lead to displacement of the native species through competition for food resources. This suggests that impacts of invader species against native species are expected to be more severe when interspecific competition is exacerbated by temperature rises.


Introduction
Global warming has been increasing at an accelerated rate [1] and seriously having an impact on various organisms and ecosystems [2][3][4][5].Especially, the poleward expansion or migration of species that originated in low latitudes is a well-known consequence of the warming [6][7][8].For example, temperature increases have facilitated the invasion of a dengue mosquito in northern Italy [9], expanded poleward the geographical distribution of dragonflies in the UK [10], and it is predicted that coral habitats will extend poleward over several hundred kilometres during this century [11].However, biological invasions and expansions owing to warming occur not only horizontally, i.e. in latitude, but also vertically, in altitude [8,12], and may severely impact on biological interactions in the invaded regions [13,14].
In general, species have climatic adaptations to their native habitat ranges [15,16].Thus, invader species that originally evolved in warm areas would be more adaptable to heat than native species in colder regions [17,18].In this case, the invaders might have a competitive advantage over the native species when exposed to increasing temperatures.For example, indirect interspecific competition for resources (i.e.exploitation competition) has been implied between plants of lowland mountain areas that migrate in altitude and compete with native alpine plants as the temperature rises owing to warming, subsequently resulting in species displacement [19,20].Previous studies on competition attributed to warming between invaders and native species have mostly focused on plant species [14,[19][20][21], as factors affecting the competition are relatively easy to detect because, unlike animals, plants do not move and their habitat requirements are fewer.The main competition among plants would be for natural resources such as light, water and nutrients [22].By contrast, animal competition has more complex mechanisms, including not only indirect competition for food resources (e.g.scramble for food) but also direct fighting or attack (interference competition) between invading and native species [23][24][25][26][27][28].Therefore, animal invasions are likely to have more severe ecological impacts on local communities and ecosystems owing to the intensification of competition not just by exploitation of resources but also by interfering directly with native species.The influence of climate warming on these two types of competition between invaders and native species has not been assessed yet, despite awareness of the importance of such impacts.
Among animal invader species, dragonflies constitute a taxon known for their long-range movement [29,30].In fact, some species like the crimson marsh glider (Trithemis aurora Burmeister) have expanded poleward owing to recent warming [31,32].Trithemis aurora, which is native to Southeast Asia and Taiwan [33], was first detected in Yaeyama and Okinawa Islands of southwest Japan around the 1980s, and then moved towards temperate latitudes in the Shikoku district by the late 2000s.Furthermore, distribution of T. aurora extended to the central Kinki district (Nara Prefecture) in 2020 (figure 1).By contrast, the Japanese white-tailed skimmer (Orthetrum albistylum speciosum Selys), which shares similar ecological characteristics with T. aurora, at least at the nymphal stage, is the most common and abundant native dragonfly in Japan.Both dragonfly species ordinarily occur among the vegetation and surface layer of the muddy soil of lentic environments, including rice paddies in Japan [37,38] and thus, can frequently be collected at the same sites.It is also known that dragonfly nymphs of these two species have a sharp dynamic hook at the tip of the lower lip and they prefer similar prey [38,39].Dragonfly nymphs are important predators in aquatic ecosystems and often become top predators in habitats without fishes [40,41].Furthermore, both dragonflies belong to the same family (Libellulidae), have similar body size and growth period, and this can cause interspecific competition, especially during their nymphal stage [42,43].Competition for resources among dragonfly nymphs can indirectly affect their foraging and mortality [44,45], and the possibility that they can attack and prey on each other cannot be denied [46].Naturally, their foraging intake may also change as a result of being attacked.If the interspecific competition between both species is severe owing to these two aspects of competition (i.e.exploitation and interference), it is envisaged that the effect of warming on T. aurora may aggravate the already negative impact the invader has on O. albistylum speciosum populations and lead to competitive displacement of the native species.
royalsocietypublishing.org/journal/rsos R. Soc.Open Sci.10: 230449 In this paper, we intend to answer the following two questions: (i) does foraging intake of the invader T. aurora and native O. albistylum speciosum dragonfly nymphs change in response to increasing temperature?and (ii) is the foraging behaviour of the two species modified owing to interspecific competition when facing a resource competitor?

Information on dragonfly species tested and their characteristics
Orthetrum albistylum speciosum is a native species in central Kinki district (Nara Prefecture), a region that T. aurora has only recently invaded.These species were selected to predict threats of biological invasions in the near future.
Life-history characteristics of the two dragonflies species are as follows: O. albistylum speciosum is known to be multivoltine, and T. aurora can be either multivoltine or univoltine.In addition, although there is no evidence from the literature that either species overwinter as eggs, it is known that their nymphal stages overwinter [47].

Collection and breeding of test species
On 5 July 2021, adult females of O. albistylum speciosum were sampled from the campus of Kindai University, Nara (34°40 0 22 00 N, 135°43 0 47 00 E).On 12 July 2021, adult females of non-native T. aurora were sampled from Kaiyo town, Tokushima (east Shikoku) (33°37 0 07 00 N, 134°22 0 27 00 E), which is the northernmost point of its established range in Japan.After collection, the tip of the abdomen was inserted in a centrifuge tube filled with dechlorinated tap water (rearing water) to induce the production of eggs.
Eggs of both species were reared separately in plastic cups (66 mm diameter × 36 mm depth) containing 40 ml of rearing water, and the nymphs that emerged from the eggs were used in this  [34,35], and then collected in Okinawa Island in 1983 [34], Amami-Oshima Island in 1988 [33], and the southern Kyushu district in 1999 [34].Its distribution had spread to the Shikoku district by the late 2000s [33,36].Furthermore, distribution of T. aurora extended to the central Kinki district (Nara Prefecture) in 2020.Solid blue and red circles show native of T. aurora and its invaded regions, respectively.The solid green circle indicates the latest area of T. aurora detection (Nara Prefecture, central Kinki district).
royalsocietypublishing.org/journal/rsos R. Soc.Open Sci.10: 230449 experiment.Eggs and nymphs were kept in an incubator (LH-30-8CT, Nippon Medical & Chemical Instruments Co, Ltd., Osaka, Japan) that controlled the following three temperatures tested in separate treatments: 27, 29 and 31°C.The lowest temperature (27°C) represents the control, and was chosen as the 20-year average temperature of the warmest month during the activity period of O. albistylum speciosum nymphs at the collection site (https://www.data.jma.go.jp/obd/stats/etrn/index.php).The other two treatments follow the global warming scenarios set by the Intergovernmental Panel on Climate Change AR6 [1], which indicate increases of +2°C (SSP1-2.6)and +4°C (SSP5-8.5)with respect to the control.By contrast, the warmest monthly average temperature during the activity period of T. aurora in its native region (Taiwan) was about 31°C during the past decade (https://www.cwb.gov.tw/V8/E/C/Statistics/monthlydata.html), which is the same as the highest treatment in our experiment.Also, an incubator photoperiod was set at 14 L : 10 D hours with reference to the sunlight hours of the warmest month in the collection site of O. albistylum speciosum nymphs, and this setting was based on data from the National Astronomical Observatory of Japan (https://eco.mtk.nao.ac.jp/ koyomi/dni/2021/s3008.html).Nymphs of both species hatched within 6-10 days after eggs were collected, and then reared individually in plastic cups (66 mm diameter × 36 mm depth).Nymphs of both species were fed nauplius larvae of Artemia salina L. (tetra brine shrimp eggs, Spectrum Brands Japan, Inc, Kanagawa, Japan) once every second day.

Foraging tests
To verify effects of an invading competitor on the foraging behaviour of native species, predation experiments of T. aurora and O. albistylum speciosum nymphs were performed using the method of De Block et al. [48] and Inouye [49] with some modifications.To remove the effect of time and/or photoperiod, all foraging tests were conducted only during the daytime when the light in the incubator was turned on (i.e.10.00 to 18.00).Foraging intakes of A. salina nauplius larvae were firstly tested on each species under the situation without a competitor (stand-alone test).Test procedures are as follows: (i) for each temperature treatment and species, 9-10 replicates were used, each replicate consisting of a plastic cup (see above) that contained one individual; (ii) nymphs about two-months old after hatching (about 4-8 mm in size) were selected randomly and transferred to plastic cups containing 40 ml of rearing water; (iii) after 1 h, thirty A. salina larvae per cup were introduced, and then the experiment was started; and (iv) the number of A. salina remaining 2 h after the experiments was counted to estimate the maximum foraging intake of each species.
To examine changes in the foraging intake of each species attributed to the presence/absence of a competitor, an experiment was conducted under the condition that both species were present in a cup (cohabitation test) with reference to additive designs which can detect a significant effect of interspecific competition on the performance of a focal species [49], and the results of this test were compared to the results of the stand-alone test.Procedures were almost the same as in the stand-alone experiment, the main difference being that each cup contained two nymphs randomly paired, one of each species.To remove effects of differences among individuals, this experiment used the same individuals as those used in the stand-alone tests.In the cohabitation experiment, however, 60 A. salina larvae were introduced into the plastic cups to account for the two nymphs present.This design enabled us to discern the effects of interference competition from the exploitation competition that would result from a decrease in available food.
To determine the effect of temperature on the competitive interference between dragonflies, we counted the number of attacks among the two species.Both foraging intake and attacks were observed during 2 h in each experiment.Foraging intake was defined as the number of A. salina eaten per nymph.An attack was defined as making contact with the other species and having the touched individual turned its head away from the attacker.When one competitor preyed on the other, data on the foraging intakes were excluded from analyses.
To understand the effect of body size on the competition, the body length as an indicator of body sizes (length from the head without antenna to the abdomen including caudal appendages) of all individuals tested were measured before the experiments.

Statistical analysis
To test effects of temperature increases and the presence/absence of a competitor on foraging intakes of each species, we used generalized linear mixed models (GLMMs; with Poisson errors and log link), where the foraging intake of O. albistylum speciosum or T. aurora nymphs was the response variable in royalsocietypublishing.org/journal/rsos R. Soc.Open Sci.10: 230449 the stand-alone and cohabitation tests.The explanatory variables were as follows: temperature, presence/ absence (p/a) of a competitor, body size and their interactions (temperature × competitor, body size × competitor).Also, individual differences in each dragonfly nymph tested were fitted as random effects.Replicates in which predation from a competitor occurred during the monitoring period were excluded from the analysis.Furthermore, relationships between the number of attacks from a competitor during the experiments and temperatures were analysed using generalized linear models (GLMs; with Poisson errors and log link), with the number of attacks from each species as the response variable and temperatures as the explanatory variable in the cohabitation test.All statistical analyses were conducted using R v. 4.1.3[50].GLMs and GLMMs were conducted using the package 'glmmTMB' [51].

Results
Although the foraging intake of O. albistylum speciosum nymphs did not differ among temperatures in the stand-alone test, the intake of this species significantly decreased with increasing temperatures when T. aurora was present (temperature: z = −1.027,p = 0.305; temperature × p/a of a competitor: z = −2.318,royalsocietypublishing.org/journal/rsos R. Soc.Open Sci.10: 230449 p = 0.021; figure 2a and table 1).The number of attacks by T. aurora on the native species increased significantly with increasing temperatures (z = 4.460, p < 0.001; figure 2b).By contrast, the foraging intake of T. aurora significantly increased with increasing temperature irrespective of the presence of a competitor or not (temperature: z = 2.079, p = 0.038; temperature × p/a of a competitor: z = 1.078, p = 0.281; figure 2c and table 2).Similarly, the number of attacks from O. albistylum speciosum on the invader increased significantly with increasing temperatures (z = 3.487, p < 0.001; figure 2d ).The foraging intake of O. albistylum speciosum did not change regardless of its body size in the standalone test, whereas the intake significantly decreased in the smaller individuals when tested under the cohabitation condition (body size: z = −0.156,p = 0.876; body size × p/a of a competitor: z = 2.352, p = 0.019; figure 3a and table 1).However, T. aurora had similar foraging intake irrespective of its body size and the presence or absence of the native competitor (body size: z = −1.156,p = 0.248; body size × p/a of a competitor: z = −0.043,p = 0.966; figure 3b and table 2).Furthermore, O. albistylum speciosum nymphs were preyed upon by T. aurora in all temperatures tested (three individuals at 27/29°C, one at 31°C).

Discussion
Our study provides a new perspective about the threats that biological invasive species pose to native species via interference competition along with warming.These two components have rarely been discussed, and we have shown that ecological impacts can be more severe with rising temperatures.As we expected, the foraging behaviour of the native dragonfly species (O.albistylum speciosum) was negatively affected and resulted in a decrease in foraging intake through encounters with the invading species (T.aurora).Although the foraging intake of O. albistylum speciosum in situations without a competitor was similar irrespective of the temperature, such intake significantly decreased with increasing temperatures when the invader T. aurora was present (figure 2a).This indicates that adverse impacts of invaders on native species via food acquisition may become more severe with current and future warming of the planet.
The number of attacks by T. aurora on the native species clearly increased with temperature (figure 2b).This phenomenon may be associated with better performance of T. aurora under warmer conditions because this species inhabits subtropical zones and is naturally adapted to such conditions.Given that the resource acquisition rate is related to the survival rate of a species [52], our results support previous studies showing that survival rates of invaders from warmer regions can be higher when temperature rises [17].Since the attacking behaviour of T. aurora is an important factor which directly diminishes the feeding ability of O. albistylum speciosum, this native species will be exposed to further adverse effects in terms of food acquisition when the temperature rises in the presence of the invader competitor, and this combined effect will inevitably lead to a decrease of its population.In addition, while the stand-alone experiment demonstrated that the foraging intake of O. albistylum speciosum nymphs remains unaltered irrespective of its body size, the smaller nymphs experienced higher foraging loses when confronted with the invader T. aurora (figure 3).This suggests that the behaviour of T. aurora was more effective against the smaller nymphs of O. albistylum speciosum, which were outcompeted by the invader.In response to these findings, we tested the relationship between the body size of O. albistylum speciosum and the body size difference of O. albistylum speciosum and T. aurora.The result showed a significant relationship between the two factors (r = −0.81,t 18 = −5.86,p < 0.001; Pearson's correlation test; electronic supplementary material, figure S1).In other words, O. albistylum speciosum individuals, which are relatively smaller than T. aurora, are exposed to a stronger impact in relation to foraging ability.Conversely, the larger O. albistylum speciosum individuals will have a smaller impact.This suggests a size-mediated priority effect [53,54].By contrast, although O. albistylum speciosum clearly increased its attacks towards the invader under situations of higher temperatures (figure 2d), the foraging intake of T. aurora increased regardless of the existence of a competitor (figure 2c), thus suggesting the attacking behaviour of the native did not impact on the foraging ability of the invader.In other words, the foraging intake of O. albistylum speciosum is negatively affected by T. aurora invasion under higher temperature conditions, but not vice versa.Given that both types of competition, interference and exploitation probably occur among dragonfly nymphs in resource-limited environments [45], the increasing fitness of T. aurora under warm conditions suggests this species is a real threat towards native dragonflies owing to global warming.Indeed, expected temperature rises may intensify the interspecific competition among dragonflies owing to both exploitation and interference competition.Moreover, the unilateral predation of T. aurora upon O. albistylum speciosum can be described as asymmetrical intraguild predation, which is one of the mechanisms causing species displacement [55], since this predation was observed in all temperatures in cohabitation tests despite feeding the nymphs saturated amounts of food.
Our primary objective was to quantify the effects of invasive competitors attributed to warming on the food acquisition performance of native species, not to compare the relative strength of its inter-and intraspecific competitive effects.The observed asymmetric interspecific competition between T. aurora and O. albistylum speciosum under increasing temperatures may result in T. aurora's competitive advantage, which could eventually lead to the displacement of the native O. albistylum speciosum in Japan.It should be noted that the effects of intraspecific competition are not considered in this Fitted curves and bands for 95% confidence interval with GLMMs.The asterisks indicate that the interaction between temperature and the presence or the absence of competing species is significant at p < 0.05 and n.s.indicates no significant difference.
royalsocietypublishing.org/journal/rsos R. Soc.Open Sci.10: 230449 experiment.To properly predict the outcomes of competitive coexistence (or displacement), both intraand interspecific interaction parameters are needed.Moreover, in the real world, O. albistylum speciosum would compete not only with T. aurora, but also with other dragonfly species and also be exposed to antagonistic interactions with top predators such as fishes [54].The presence of such 'third species' may modify the interaction between T. aurora and O. albistylum speciosum, which makes it more difficult to predict the outcomes of T. aurora invasion.Thus, for properly extrapolating our experimental results to more complex field environments and predicting more accurately the ecological threats of T. aurora invasion we need to consider these perspectives comprehensively in future studies.

Conclusion
Our results support the hypothesis of the intensification of interference competition between invading and native species with increasing temperatures.This implies that impacts of biological invasions on native species through direct attacks might be exacerbated by global warming.Consequently, the impact of invader species from warmer regions on native species may be more severe than previous studies suggested because that competition for resources intensifies as the ambient temperate rises [19,20].Our study proved this is the case for the two dragonfly species considered here.Thus, the possibility of a species displacement by a poleward-expanding invader through the two types of competitions (exploitation and interference) is real.However, our experiments were conducted within plastic cups containing only rearing water without any substrates (e.g.plants, mud and rock) that would allow the two dragonfly nymphs to hide.For instance, nymphs of Aeshna viridis Eversmann tend to avoid the intraguild predation by using vegetation as a refuge [56].As mentioned before, O. albistylum speciosum nymphs often inhabit the vegetation or surface layer of the muddy soil in lentic environments [38], implying that they may avoid attacks from T. aurora by hiding.In addition, because in our study the two dragonfly nymphs tested were reared in the laboratory, the amount of food provided to these nymphs might have not been sufficient.Therefore, it is possible that the feeding frequency would have affected the competitive interactions, resulting in a different outcome to what occurs in the real world.To validate this prediction, it is important to assess not only interspecific competition between the two dragonfly nymphs but also competition with other species under experimental test systems that mimic the field environment (e.g.mesocosms).How the impact posed by biological invasions to ecosystems change with warming is an important global issue, but it is very difficult to predict whether the impact will be severe or mild owing to the complexity of its process and mechanisms (e.g.[57]).On the other hand, our finding that 'threats by poleward-expanding species that invade from warmer regions become severe with warming' would be applied to other cases of biological invasions attributed to global warming.This is because poleward-expanding invaders originally inhabit warmer regions and thus, a further warming is expected to bring the invaded area closer to the temperature of their origin.
Recently, poleward-expanding invaders have been reported in various taxonomic groups and the importance of predicting their impacts is growing [6][7][8][9][10][11][12]17,18,58,59].Our viewpoint should be applied to predict impacts of new invaders attributed to warming regardless of taxonomic groups.In summary, threats of biological invasions associated with warming are expected to be more severe than the effects of biological invasions alone owing to unbalanced interspecific competition through both exploitation and interference.

Figure 1 .
Figure 1.Tendency of the distribution expansion of the invasive Trithemis aurora in Japan.Trithemis aurora, native to Southeast Asia and Taiwan (subtropical to tropical climates), was first detected in subtropical Ishigaki Island (southwest Japan) in 1981[34,35], and then collected in Okinawa Island in 1983[34], Amami-Oshima Island in 1988[33], and the southern Kyushu district in 1999[34].Its distribution had spread to the Shikoku district by the late 2000s[33,36].Furthermore, distribution of T. aurora extended to the central Kinki district (Nara Prefecture) in 2020.Solid blue and red circles show native of T. aurora and its invaded regions, respectively.The solid green circle indicates the latest area of T. aurora detection (Nara Prefecture, central Kinki district).

30 AFigure 2 .
Figure 2. A. Effects of the invading competitor T. aurora (Ta) on native O. albistylum speciosum (Oas).(a) Effect of temperature and presence of the invader on the foraging intake of the native species under two experimental conditions: stand-alone (blue), and cohabitation (purple); (b) number of attacks of T. aurora on O. albistylum speciosum.B. Effects of a native competitor on invading T. aurora.(c) Effects of temperature and presence or absence of Oas on the foraging intake of Ta under the two experimental conditions: stand-alone (red) and cohabitation (purple); (d) number of attacks of O. albistylum speciosum on T. aurora.Fitted curves and bands for 95% confidence interval with GLMMs and GLMs.In (a) and (c) the asterisks indicate that the interaction between temperature and the presence or absence of competing species is significant at p < 0.05 and n.s.indicates no significant difference.

Figure 3 .
Figure 3. (a) Effect of the body size and the presence or the absence of T. aurora on the foraging intake of O. albistylum speciosum under the two experimental conditions: stand-alone (blue) and cohabitation (purple).(b) Effect of the body size and presence of O.albistylum speciosum on the foraging intake of T. aurora under the same two conditions: stand-alone (red) and cohabitation (purple).Fitted curves and bands for 95% confidence interval with GLMMs.The asterisks indicate that the interaction between temperature and the presence or the absence of competing species is significant at p < 0.05 and n.s.indicates no significant difference.

Table 2 .
Effects of temperature, presence/absence (p/a) of a competitor and body size on foraging intakes of Trithemis aurora nymph using GLMMs ( Ã p < 0.05).